Novel process of producing 1, 3, 4, 10, 11, 12-hexahydroxy-6-methylnaphthacene-2-carboxamide



United States Patent 3,3s5,7ss NOVEL PROCESS OF PRODUCING1,3,4,10,11,12- HEXAHYDROXY 6 METHYLNAPHTHACENE- Z-CARBOXAMIDE JerryRobert Daniel McCormick, Spring Valley, Ursula Joachim, White Plains,and Elmer Raymond Jensen, Nanuet, N.Y., and Newell Oscar Sjolander,Saddle River, N.J., assignors to American Cyanamid Company, Stamford,Conn, a corporation of Maine No Drawing. Filed Feb. 3, 1965, Ser. No.430,184 3 Claims. (Cl. 19580) ABSTRACT OF THE DISCLOSURE l,3,4,l0,11,12hexahydroxy 6-methylnaphthacene-2- carboxamide and6,ll-dehydro-l1-keto-6-methyl-3,6,l0, 12tetrahydroxynaphthacene-1,4-quinone-2-carboxamide are produced andrecovered from the submerged aerobic fermentation of blocked mutants ofStreptomyces aureofacicns.

This invention relates to a novel process of producing l,3,4,l0,ll,12hexahydroxy 6 methylnaphthacene 2- carboxamide and, more particularly,is concerned with the production of1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-2-ca1'boxamide by thesubmerged aerobic fermentation of certain blocked mutants ofStreptomyces aureofaciens.

The present invention is based upon the discovery that selected strainsof S. am-eofaciens are capable of the simultaneous fermentativebiosynthesis of a major proportion of1,3,4,10,ll,12-hexahydroxy-G-methylnaphthacene-2-carboxamide togetherwith a lesser proportion of 6,11 dihydrol1-keto-6-methyl-3,6,10,12-tetrahydroxynaphthacene-1,4-quinone-2-carboxamideas a by-product.

Strains useful for this purpose can be recognized by the green color ofcolonies on corn steep liquor-containing agar, and by the green color offermentation harvest mashes when grown in corn steep liquor-containingliquid media. These strains can be further characterized by theappearance of a broad ultraviolet absorption maximum peaking at 450-470millimicrons when the ultraviolet absorption spectrum is run on a 0.2 Nhydrochloric acid-indimethylformamide extract of the whole harvest mash.This is accomplished by diluting ml. of the whole harvest mash to 50 ml.with a 0.2 N solution of hydrogen chloride in dimethylformamide, andthen filtering through No. 12 Whatman filter paper. Readings are made onthe filtrate in a one-centimeter cell in a Cary RecordingSpectrophotometer, Model 11. If necessary, further dilution of thefiltrate with a 0.1 N solution of hydrogen chloride in dimethylformamidemay be made in order to give spectrophotometric absorbency readingsbetween 0.25 and 1.50 in the region of 450-470 m maxima.

A number of strains which produce these two compounds have been obtainedby selection from, and mutation of, S. aureofaciens parental strains,such parental strains being characterized by the ability to produceeither tetracycline or 7-chlorotetracycline. Representative strainswhich may be used to practice this invention are S. aureofaczens 8730-6,V1383, V655 and 8294-4. The latter two Patented May 28, 1968 strainshave been deposited as NRRL 3132 at the Northern Regional ResearchLaboratories, Peoria, 111., and as ATCC 1076211 at the American TypeCulture Collection, Washington, DC, respectively, Further operativestrains may be readily found by the usual methods of strain selection.

The conditions of the fermentation are generally the same as thepresently known methods of producing tetracycline, 7-chlorotetracyclineor S-hydroxytetracycline by fermentation. That is, the fermentationmedium contains the usual nutrients and mineral substances. Suitablenutrient substances include starch, dextrose, cane sugar, glucose,molasses, soybean meal, peanut meal, yeast, meat extracts, peptone,ammonium sulfate, u-rea, corn steep liquor, distillers solubles, wheatgluten, cottonseed meal, inorganic salts and other conventionalsubstances. The inorganic salts include calcium carbonate, ammoniumsulfate, ammonium chloride and the various trace elements such asmanganese, cobalt, zinc, copper, iron and the like.

The other general conditions of the fermentation, such as hydrogen ionconcentration, temperature, time, rate of aeration, alternative methodsfor preparation of the inoculum, sterilization, inoculation and the likeare conventional and may be similar to those conditions used for theproduction of 7-chlorotetracycline shown in U.S. Patent No. 2,482,055 toDuggar; for the production of tetracycline shown in US. Patent No.2,734,018 to Minieri et a1. and for the production ofS-hydroxytetracycline shown in US. Patent N0. 2,516,080 to Sobin et al.

A suitable inoculum may be obtained by employing spores of the properstrain of S. aureofacz'ens, propagated on agar slants or Roux bottles,to inoculate a nutrient medium contained in shaker flasks or bottles.After incubation, this inoculum is transferred to a larger volume ofnutrient medium in a seed tank. After allowing sufiicient time for theculture to develop, the seed tank growth is transferred to a fermentor,under aseptic conditions, and the fermentation is continued for asuitable period of time.

The recovery of the 1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-2-carboxamide from the whole harvest mash may beachieved by extraction with tetrahydrofuran. The6,1l-dihydro-l1-ket0-6methyl-3,6,l0,12-tetrahydroxynaphthacene-l,4-quinone-2carboxamide remains in the mother liquor and may be isolated either byevaporation or by dilution and precipitation with a non-solvent.

The compound 6,11-dihydro-ll-keto-6-rnethyl-3,6,10, 12tetrahydroxynaphthacene-1,4-quinone-Z-carboxamide is a crystallineproduct which dissolves in alkali with an emerald green color and whichhas the following characteristic absorption maxima:

X355; H2SO41% boric acid, 275 (shoulder), 310, 472 (shoulder), 535.

This compound, 6,1l-dihydro-l1-keto-6-methyl-3,6,10,12-tet-rahydroxynaphthacene-1,4-quinone-2-carboxamide, is a new compoundand its utility lies in the fact that it may be converted to1,3,4,10,l1,12 hexahydroxy 6 methylnaphthacene-Z-carboxamide by heatingin a solvent such as phenol with an acidic reducing agent such ashydriodic acid, tinhydrochloric acid, or hypophosphorous acid.

The 1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene- 2-carboxamide isuseful as an intermediate in the synthe- 3 sis of physiologically activeantibiotics of the tetracycline series. For example,1,3,4,10,11,l2-hexahydroxy-6-methylnaphthacene-Z-carboxamide may bebiologically transformed to tetracyclines as set forth in the followingreaction scheme:

wherein R is hydrogen or hydroxy and R is hydrogen, chlorine or bromine.This transformation (which in its simplest form consists of the netaddition of two moles of Water, one at 4a,l2a and one at 5a,6a; and thereplacement of the 4-hydroxyl group by a dimethylamino group) isaccomplished by adding 1,3,4,l0,l1,12-hexahydroxy-6- methylnaphthacene 2carboxamide to a fermentation medium inoculated with a strain of aspecies of the genus Streptomyces, which species is capable of producingone of the tetracyclines. Certain other biological transformations maybe accomplished simultaneously with the 5a,6aand 4a,l2a-dihydration andthe introduction of the 4-dimethylamino group. Where a S-hydroxyl-atingspecies of the genus Streptomyces is employed, then a hydroxyl group isintroduced at the 5-position. Where a halogenating strain of the genusStreptomyces is employed, then R, is chlorine or bromine depending uponthe conditions of the fermentation. Among the strains of S. aureoyacienswhich will introduce chlorine or bromine at the 7-position of themolecule are the following:

S. aureofaciens ATCC 107620 ATCC 13189 ATCC 10762g ATCC 13899 ATCC10762: ATCC 13900 ATCC 11989 NRRL B-1286 ATCC 12416b NRRL B1287 ATCC124160 NRRL B-1288 ATCC 124l6d NRRL B-2209 ATCC 12551 NRRL B-2406 ATCC12552 NRRL B-2407 ATCC 12553 NRRL 3013 ATCC 12554 A- representativestrain of the genus Streptomyces which is a non-halogenating strain,that is, which will not introduce halogen at the 7-position of themolecule, is S. aureofaciens NRRL 3014. Representative strains of thegenus Streptomyces which are non-halogenating strains but which willintroduce a hydroxy group at the 5-position of the molecule, are S.rimosus NRRL 2234, S. platenis NRRL 2364 and S. hygrosc-opicus NRRL3015.

The conditions of the fermentation for the biological conversion of1,3,4,10,11,12-hexahydroxy-6-methyln-aphthacene-Z-carboxamide totetracyclines are generally the same as set forth in US. Patent2,482,055 to Duggar, US. Patent 2,734,018 to Minieri et al. and US.Patent 2,878,289 to McCormick et al. and which, in turn, are generallythe same as for the presently known methods for producing varioustetracyclines by fermentation. That is, the fermentation medium containsthe usual nutrients and mineral substances. Suitable nutrients includeany assimilable source of carbon, such as the polysaccharides orstarches, or polyalcohols such as glycerol may be used. An assirnilablesource of nitrogen may be supplied through the use of proteins, proteinhydrolysates, urea, corn steep liquor, meat extracts, peptone,distillers solubles, fish meal and other conventional substances. Thecommon essential anions and cations are supplied in the form of theirnontoxic salts. Trace elements such as manganese, cobalt, Zinc, copper,etc., are obtained either as impurities in the above compounds, orthrough the use of tap water or by specifically adding solutionsespecially enriched with these trace elements.

The other general conditions of the fermentation such as hydrogen ionconcentration, temperature, time, rate of aeration, preparation of theinoculum, sterilization, inoculation and the like are conventional andare similar to those for the production of other tetracyclines as setforth in the aforementioned US. Patents to Duggar, Minieri et al. andMcCormick et al.

When a 7-halogenating strain of the genus Streptomyces is employed with1,3,4,10,1l,l2-hexahydroxy-6-methylnaphthacene-Z-carboxamide, it isnecessary only to modify the fermentation medium so that it contains atleast 10 parts per million of chloride ions when the 7-chlorosubstituent is desired, or alike amount of bromide ions when the 7-bromosubstituent is desired.

After the fermentation has been continued for a suitable time, forexample, from 12 to 96 hours, and the transformation of 1,3,4,l0,11,l2hexadroxy 6 methylnapththacene-Z-carboxamide to the desired tetracyclineis substantially complete, the tetracycline product may be isolated fromthe fermentation mash in any convenient manner. The isolation processmay be selected from any of the numerous isolation techniques now wellknown in the art.

The starting material may be added at any desired concentration,although for practical reasons a substrate at a concentration of up toabout 10 grams per liter of medium is satisfactory although higherconcentrations may be used with some sacrifice in yield. The addition ofthe starting material may be accomplished in any suitable manner so longas it promotes contact with the biological medium. To this end, it ispreferred to add the starting material in a solvent such asdimethylformamide, dimethylacetamide, dimethylsulfoxide,tetramethylenesulfoxide and N-methylpyrrolidone. However,dimethylsulfoxide is preferred and a solution of magnesium acetate indimethylsulfoxide is the most preferred solvent for the startingmaterial. Solutions of the1,3,4,l0,l1,l2-hexahydroxy-6-methylnaphthacene-Z-carboxamide must beprotected from air as the compounds "are readily oxidized in solution.

The invention will be described in greater detail in conjunction withthe following specific examples.

EXAMPLE 1 Preparation of 1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-Z-carboxamide Grams Sucrose 30 Ammonium sulfate 2Calcium carbonate 7 Cornsteep liquor 20 Tap water, qs. to 1000milliliters.

Prior to inoculation, the medium was sterilized by autoclaving for 20minutes under a pressure of 15 pounds per square inch. The inoculatedtube was then incubated for 24 hours at 28 C. on a reciprocating shakeroperating at 116 oscillations per minute, whereby an inoculum of the S.aureofaciens was obtained. A fermentation medium of the followingcomposition was prepared:

Cottonseed flour "grams" 35.0 Corn starch do 50.0 Calcium carbonate do8.0 Yeast do 1.0 Ammonium chloride do 1.0 Copper sulfate do 0.05 Lardoil, v./v. percent 3.2

Tap water, qs. to 1000 milliliters.

After sterilization of this medium in an autoclave for 20 minutes at apressure of pounds per square inch, 25 ml. portions in 250 ml.Erlenmeyer flasks were inoculated with 1.0 ml. portions of the S.ameofaciens inoculum. The fermentation was carried out for 120 hours at25 C. on a rotary shaker operating at 180 revolutions er minute. Thecontents of 35 of these flasks were pooled and killed by pasteurization.The neutral mash solids were collected by centrifugation andfreeze-dried. A 5 gram portion of this freeze-dried mash was washed on afunnel four times with 25 ml. portions of ethyl ether. The 1,3,4,10,11,12-hexahydroxy 6 methylnaphthacene-Z-carboxamide was extracted asfollows: The ether-washed solids were extracted with a 100 ml. portionof tetrahydrofuran for minutes on a rotary shaker and the extract wasfiltered. A second 20 minute extraction with a 50 ml. portion oftetrahydrofuran was carried out on the rotary shaker and the extract wasfiltered. The filtered extracts were pooled and evaporated to 5 ml.,whereupon crystallization of the1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-Z-carboxamide occurred.These crystals were isolated by filtration, washed with ethyl ether andthen air dried. The filtrate and washings from this isolation werepooled and evaporated to dryness. To the resulting crude6,1l-dihydro-l1-keto-6-methyl3,6-10,12-tetrahydroxynapthacene-1,4-quinone-2-carboxamide were added 2ml. of phenol, 1 ml. of hydriodic acid and 0.1 gram of potassiumhypophosphite in a test tube. The mixture was heated to reflux for 5minutes and then cooled to room temperature. The orange, crystalline,1,3,4,l0,11,12-hexahydroxy 6 methylnaphthacene-Z-carboxamide whichformed was collected by filtration and air dried. The total yield of1,3,4,l0,11,12 hexahydroxy-6-methylnaphthacene-Z-carboxamide was 46milligrams.

EXAMPLE 2 Conversion of1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-Z-carboxamide to7-chlorotetracycline Spores of S. aureofaciens NRRL 3013 were washedfrom an agar slant with sterile distilled water to form a suspensioncontaining 60 million to 80 million spores per ml. A 0.33 ml. portion ofthis suspension was used to inoculate an 8 inch test tube containing 8ml. of an inoculum prepared as described in Example 1. A fermentationmedium of the following composition was prepared:

(NH S0 grams 6.7 CaCO do 9.0 CoCl -6H O mil1igrams 5.0 NH Cl grams 2.0MnSO (70% technical grade) ..dO 0.10 Cornsteep liquor do 25.0 Starch d052.5 Corn flour -do 14.5

Tap water, qs. to 1000 milliliters.

After sterilization of this medium, ml. portions in 250 ml. Erlenmeyerflasks were inoculated with 1 ml. portions of the S. aureofaciens NRRL3013 inoculum. The fermentation was carried out at 28 C. for 24 hours ona rotary shaker operating at 180 revolutions per minute. At this timeeach mash portion was transferred to an individual flask containing asolution of 10.7 mg. of 1,3,4,10,11,12-hexhahydroxy-6-methylnaphthacene-Z-carboxamide in a mixture of 30 mg. ofmagnesium acetate and 1 ml. of dimethylsulfoxide. The fermentation wascontinued on the rotary shaker for an adidtiona-l 96 hours at 28 C. Atthis time, biological assays of the mash indicated the presence ofantibacterial activity corresponding to 264 micrograms of7-chlorotetracycline per ml. This corresponds to a yield of 49% based onthe compound added. The identity of the product as 7-chlorotetracyclinewas confirmed by paper chromatography in a butanol-pH3 phosphate buflersystem. A control flask run in the same manner but with the addition ofonly 30 mg. of magnesium acetate and 1 ml. of dimethylsulfoxide and no1,3,4,10,11,12- hexahydroxy 6 methylnaphthacene 2 carboxamide, showed no7-chlorotetracycline.

EXAMPLE 3 Large scale preparation of 1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-2-carboxamide and conversion to l-chlorotetracyclineSpores of S. aureofaciens NRRL 3132 were washed from an agar slant toform a suspension containing 60 million to million spores per ml. A 2.0ml. portion of this suspension was used to inoculate 15 ml. of a mediumprepared according to the following formulation:

Grams per liter Cornsteep liquor 30 Starch 30 Calcium carbonate 10Ammonium sulfate 2 Prior to inoculation the medium was sterilized byauto- Starch grams per liter 40 Cottonseed flour do 35 CoCO do 6 Yeastdo 1 NH Cl do 1 CuSO -5H O milligrams per liter 50 Lard oil, v./v.percent 3 After sterilization of this medium, a 7.5 liter portion in a12 liter bottle was inoculated with the 375 ml. portion of S.aureofaciens inoculum. The fermentation was carried out at 25 C. forhours with mechanical stirring and aeration with sterile air.

A 15 ml. portion of the heat-skilled harvest mash in a mixture ofappropriate quantities of magnesium acetate and dimethylsulfoxide wasadded to 25 ml. of a 24 hour fermentation mash of S. aureofaciens NRRL3013 and the fermentation was continued as described in Example 2.Biological assay of the whole harvest mash revealed a concentration ofantibacterial activacterial activity corresponding to 187 micrograms of7-chlorotetracycline per ml. The identity of the product as7-chlorotetracycline was confirmed as described in Example 2.

What is claimed is:

1. A process for the concomitant production of 1,3,4,10,11,12-hexahydroxy 6 methylnaphthacene-Z-carboxamide and6,1l-dihydro-l1-keto-6-methyl-3,6,10,12-tetrahydroxynaphthacene-1,4-quinone2 carboxamide which comprises cultivating a blocked mutant ofSterptomyces aureofaciens in an aqueous nutrient medium containingassimilable sources of carbohydrate, nitrogen and inorganic salts undersubmerged aerobic conditions until substantial quantities of1,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-Z-carboxamide and6,11-dihydro-11-keto- 6-methyl-3,6,10,12tetrahydroxynaphthacene1,4-quinone- 2-carboxamide are produced in saidmedium, and recovering the1,3,4,l0,11,1Z-hexahydroxy-6-methylnaphthacene- 2-carboxamide and6,1l-dihydro-ll-keto-6-methy1-3,6,10, 12tetrahydroxynaphthacene-1,4quinone-2-carboxamide so-produced; saidblocked mutant of S. aureofaciens being characterized by having anabsorption maximum at 450- 470 III/J, when the ultraviolet absorptionspectrum is determined on a 0.2 N hydrochloric acid-in-dimethylformamideextract of the Whole harvest mash.

2. A process for producing 1,3,4,10,11-12-hexahydroxy-6-methylnaphthacene 2 carboxamide which comprises cultivating a blockedmutant of Streptomyces aureofaciens in an aqueous nutrient mediumcontaining assimilable sources of carbohydrate, nitrogen and inorganicsalts under submerged aerobic conditions until substantial quantities of1,3,4,10,11,12-hexahydroxy-o-methylnaphthacene-Z-carboxamide areproduced in said medium, and recovering thel,3,4,10,11,12-hexahydroxy-6-methylnaphthacene-Z-carboxamideso-produced; said blocked mutant of S. aureofaciens being characterizedby having an absorption maximum at 450-470 m when the ultravioletabsorption spectrum is determined on a 0.2 N hydrochloricacid-in-dimethylformamide extract of the whole harvest mash.

3. A process for producing 6,11'dihydro-11-keto-6- methyl-3,6,10,12tetrahydroxynaphthacene-1,4-quinone- 2-carboxamide which comprisescultivating a blocked mutant of Streptomyces aureofaciens in an aqueousnutrient medium containing an assirnilable source of carbohydrate,nitrogen and inorganic salts under submerged aerobic conditions untilsubstantial quantities of 6,11-dihydroy-11-keto-6-methyl 13,6,10,12tetrahydroxynaphthacene-1,4-quinone-2-carboxamide are produced in saidmedium, and recovering the 6,1l-dihydro-1l-keto-6- methyl-3,6,10,12tetrahydroxynaphthacene-1,4-quinone- 2-carboxamide so-produced; saidblocked mutant of S. aureofaciens being characterized by having anabsorption maximum at 450-470 me when the ultraviolet absorptionspectrum is determined on a 0.2 N hydrochloric acidin-dirnethylformamideextract of the whole harvest mash.

References Cited UNITED STATES PATENTS 3,226,305 12/1965 McCormick et a1195-80 MAURICE W. GREENSTEIN, Primary Examiner.

